Development and parentage analysis of SNP markers for Chestnut-vented Nuthatch(Sitta nagaensis)based on ddRAD-seq data

Extra-pair paternity(EPP)is commonly found in socially monogamous birds,especially in small passerine birds,and there are interspecific and intraspecific variations in the extent of EPP.The Chestnut-vented Nuthatch(Sitta nagaensis)is a socially monogamous passerine bird,and verifying whether this species has EPP relies on parentage testing-S.nagaensis is not known to have EPP.In this study,we developed SNP markers of this species that are informative for parentage analysis from double digest restriction site-associated DNA sequencing(ddRAD-seq)data.A panel consisting of 50 SNP markers,with a mean heterozygosity of 0.343,was used to resolve 95% of nestlings to fathers.The combined exclusion probabilities for the first parent and second parent were 0.991 and 0.9999,respectively.This panel of SNP markers is a powerful tool for parentage assignments in S.nagaensis.In addition,we found that three offspring(7.9%)from three nests(23.1%)were the result of extra-pair fertilization out of 38 offspring in 13 nests.Our study provided information on parentage analysis that has not been reported before in S.nagaensis.It also supplemented the understudied EPP behavior of birds in Asia,contributing to a general understanding of the EPP behaviors of birds.

Development of SNP parentage assignment techniques in the yellowfin seabream Acanthopagrus latus

Acanthopagrus latus is an essential aquaculture species on the south coast of China.However,there is a lack of systematic breeding of A.latus,which considerably limits the sustainable development of A.latus.As a result,genetic improvements are urgently needed to breed new strains of A.latus with rapid growth and strong resistance to disease.During selective breeding,it is necessary to estimate the genetic parameters of the target trait,which in turn depends on an accurate disentangled pedigree for the selective population.Therefore,it is necessary to establish the parentage assignment technique for A.latus.In this study,95 individuals selected from their parents and their 14 families were used as experimental material.SNPs were developed by genome resequencing,and highly polymorphic SNPs were screened on the basis of optimized filtering parameters.A total of 14392738 SNPs were discovered and 205 SNPs were selected for parentage assignment using the CERVUS software.In the model where the gender of the parents is known,the assignment success rate is 98.61%for the male parent,97.22%for the female parent,and 95.83%for the parent pair.In the model where the gender of the parents is unknown,the assignment success rate is 100%for a single parent and 90.28%for the parent pair.The results of this study were expected to serve as a reference for the breeding of new varieties of A.latus.

Identification of the Parentage of Corn Variety Using SSR Markers

[Objective] The study was to explore the molecular interpretation standards on parentage in the seeds of corn variety. [Method] With 16 hybrids and their parents and 202 inbred elites as materials for screening primers, the artificial groups of two standard diad and two standard triad were respectively established as the verification materials. Genomic DNA of seedlings was extracted by using CTAB method. 137 pairs of SSR primers were selected for SSR amplification and product detection, which was used for the parentage identification of maize varieties. [ Result] Twenty pairs of corn primers with high polymorphism information content （ PIC value）, clearly amplified bands and good reproducibility were screened from 137 pairs of corn SSR primers tested. The identification results of using SSR molecular were consistent with the actual situation. [ Conclusion] It is feasible to identify the parentage of maize variety using SSR markers.

Establishment of microsatellite-based triplex PCR for parentage analysis of Chinese shrimp Fenneropenaeus chinensis

Through exploring the microsatellite primers from the random genome sequences of Chinese shrimp （Fenneropenaeus chinensis）, some microsatellite primers were obtained with rich polymorphic genetic information, and a triplex PCR was established using three primers （RS1101, RS0683 and H081 primers）. By adjusting the final concentration of Mg^2＋, dNTP and primers, and using a touch-town PCR program, the optimum amplification parameters of PCR system were obtained, which could successfully amplify the three primers in a PCR reaction. In the denatured PAGE gel, the amplified DNA fragments of three primers RS1 101,RS0683 and H081 could be easily identified each other. For the triplex PCR system, the PPE （probabilities of paternity exclusion） is 0.967 9,and the DP （discrimination power） is 0.999 327.Using the triplex PCR to test ten individuals of a parentage and their parents, an individual was excluded from the parentage in all of the three microsatellite loci, which might be mixed into the parentage for some unknown reason such as factitious misplay. The triplex PCR will be of great practical value in identifying the parentages of F. chinensis.

Development of Three Multiplex PCR Primer Sets for Ark Shell(Scapharca broughtonii)and Their Validation in Parentage Assignment

Scapharca broughtonii is a commercially important and over-exploited species.In order to investigate its genetic diversity and population structure,43 novel polymorphic microsatellites were isolated and characterized.The number of alleles per locus ranged from 3 to 22 with an average of 6.93,and the observed and expected heterozygosities varied between 0.233 and 1.000,and 0.250 and 0.953,with an average of 0.614 and 0.707,respectively.Three highly informative multiplex PCRs were developed from nine of those microsatellites for S.broughtonii.We evaluated and validated these multiplex PCRs in 8 full-sib families.The average polymorphism information content(PIC) was 0.539.The frequency of null alleles was estimated as 3.13% of all the alleles segregation based on a within-family analysis of Mendelian segregation patterns.Parentage analysis of real offspring demonstrated that 100% of all offspring were unambiguously allocated to a pair of parents based on 3 multiplex sets.Those 43 microsatellite loci with high variability will be helpful for the analysis of population genetics and conservation of wild stock of S.broughtonii.The 3 sets of multiplex PCRs could be an important tool of pedigree reconstruction,population genetic analysis and brood stock management.

Multiplex PCR Sets of Novel Microsatellite Loci for Iwagaki Oyster Crassostrea nippona and Their Application in Parentage Assignment

Iwagaki oyster,Crassostrea nippona,widely distributes along the seashore of Eastern Asia,and has been considered to be a potential breeding species due to its delicious taste and high commercial value.In order to study its genetic background and population structure,we developed 46 novel polymorphic microsatellite markers using next-generation sequencing technique and characterized them in 30 individuals.The number of alleles ranged from 3 to 22,while the observed and expected heterozygosities varied from 0.133 to 1.000 and 0.455 to 0.949,respectively.Fifteen microsatellite markers were selected and grouped into five highly informative multiplex PCRs for C.nippona.We evaluated and validated these multiplex PCRs in a cultured population including 173 candidate parents and 486 offspring.In actual parentage analysis,80%of the offspring were correctly assigned to their parental pairs using three multiplex PCRs.Furthermore,the success rate of parentage assignment reached 96%when the other two multiplex PCRs were added.These 46 microsatellite loci with high variability and the five multiplex PCRs described here provide a powerful tool for pedigree reconstruction,resource conservation and selective breeding program of C.nippona.

Leading directions and effective distance of larch offspring dispersal at the upper treeline in the Northern and Polar Urals, Russia

Climate has changed sufficiently over the last 150 years and forced out upper treeline advance at the most studied sites around the world.The rate of advance has been extremely variable–from tens to hundreds meters in altitude.This is because the degree at which tree frontal populations respond to climate change depends on the complex interaction of biological and physical factors.The resulting stand pattern is the consequence of the interaction between dispersal and survival functions.A few publications have addressed the question of how this pattern is generated.In order to understand how the spatial structure of tree stands was formed at the upper limit of their distribution in the Ural Mountains,we assessed the distance and direction of dispersal of offspring from maternal individuals.We found that in frontal Larix sibirica Ledeb.populations,‘effective’dispersal of offspring ranges from 3 to 758 m(with a median of 20–33 m in open forest and 219 m in single-tree tundra in the Polar Urals and 107 m in open forest in the Northern Urals).We revealed that most of the offspring effectively dispersed not only in the direction of the prevailing winds,but also in the opposite direction up the slope,and the distance can reach 500–760 m.The data obtained can be used to develop an individual-based model which is capable of simulating in detail the dynamics of tree stands at the upper limit of their growth and reliably predicting the future position and pattern of treeline ecotone as growth conditions continue to improve in the face of observed climate change.

Parentage assignment and parental contribution analysis in large yellow croaker Larimichthys crocea using microsatellite markers

The large yellow croaker Larimichthys crocea is one of the most important fish species in China. To estimate the re- productive success of breeders, three independent full-factorial crosses were created and the fins of breeders and progenies were sampled for microsatellite analysis. Out of 959 offspring from three sets, 99.6% were assigned to their parents using 6-7 mi- crosatellite markers. In all crosses, some parent pairs produced a large number of offspring and some parent pairs did not produce any offspring. The contributions of male or female parents were unequal, ranging from 1.0-89.3% across the three sets. The loss of putative Ne was 69.6% in set 1, 31.2% in set 2 and 57.6% in set 3. These results suggest that the unequal contribution of parents is universal in artificial breeding ofL. crocea, especially in a small population, and this should be taken into account in hatcheries or when releasing animals for resource enhancement

Parentage testing anomalies in Hong Kong SAR of China

Objective To determine if there are any differences in the number of exclusions from paternity of men using an anonymous parentage testing service compared with that of men using an in-person parentage testing service provided by the same company in Hong Kong SAR of China. Methods Comparable numbers of consecutive anonymous and in-person parentage tests conducted by the same company were analyzed. Results Men using an anonymous parentage testing service were excluded from paternity at a significantly greater rate (P<0.001), compared with men using an in-person parentage test service. Conclusions The results obtained from anonymous parentage testing indicate that the number of families containing children of doubtful parentage is much greater than expected previously. As illegitimate children are known to suffer greater degrees of abuse and neglect, this finding poses serious social questions regarding the welfare of families, which the relevant authorities should acknowledge and address.

An inter-laboratory study of DNA-based identity,parentage and species testing in animal forensic genetics

The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability.Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output.The International Society for Animal Genetics(ISAG)administered animal forensic comparison tests(AFCTs)in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification,parentage and species determination services.The AFCTs revealed that analyses of low DNA template concentrations(≤300 pg/μL)constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results.Moreover,a lack of familiarity with species testing protocols,interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results.Several laboratories showed improvement in their genotyping accuracy over time.However,the use of forensically validated standards,such as a standard forensic short tandem repeat(STR)kit,preferably with an allelic ladder,and stricter guidelines for STR typing,may have prevented some common issues from occurring,such as genotyping inaccuracies,missing data,elevated stutter products and loading errors.The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other.Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel’s proficiency in critical techniques such as low copy number(LCN)analysis and species testing.Although this is the first time that the ISAG has conducted comparison tests for forensic testing,findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.

Characterization of 12 polymorphic microsatellite markers in the Chinese tree shrew (Tupaia belangeri chinensis)

The Chinese tree shrew (Tupaia belangeri chinensis) is a small experimental animal with a close affinity to primates. This species has long been proposed to be an alternative experimental animal to primates in biomedical research. Despite decades of study, there is no pure breed for this animal, and the overall genetic diversity of wild tree shrews remains largely unknown. In order to obtain a set of genetic markers for evaluating the genetic diversity of tree shrew wild populations and tracing the lineages in inbreeding populations, we developed 12 polymorphic microsatellite markers from the genomic DNA of the tree shrew. An analysis of a wild population of 117 individuals collected from the suburb of Kunming, China, showed that these loci exhibited a highly expected heterozygosity (0.616). These 12 microsatellites were sufficient for individual identification and parentage analysis. The microsatellite markers developed in this study will be of use in evaluating genetic diversity and lineage tracing for the tree shrew.

Development of two microsatellite multiplex PCR systems for high throughput genotyping in Populus euphratica

Eighteen microsatellite primer pairs previously developed at Oak Ridge National Laboratory for Populus tremuloides Michx. and Populus trichocarpa Tort. ＆ Gray were screened for amplification in Euphrates poplar, Populus euphratica Oliv. Thirteen loci were found to express polymorphisms ranging from two to 17 alleles. The eight most variable loci were selected to set up and optimize two multiplex polymerase chain reaction （PCR） assays. Three populations containing altogether 436 trees were used to characterize the selected loci and ascertain their applicability for parentage analysis and genotyping studies. Through cross-checking of clonal identity against sex of the genotyped trees we estimated the maximum error rate for merging genotypes to be less than 0.045.

Characterization of 12 polymorphic microsatellite markers in the Chinese tree shrew(Tupaia belangeri chinensis)

The Chinese tree shrew(Tupaia belangeri chinensis)is a small experimental animal with a close affinity to primates.This species has long been proposed to be an alternative experimental animal to primates in biomedical research.Despite decades of study,there is no pure breed for this animal,and the overall genetic diversity of wild tree shrews remains largely unknown.In order to obtain a set of genetic markers for evaluating the genetic diversity of tree shrew wild populations and tracing the lineages in inbreeding populations,we developed 12 polymorphic microsatellite markers from the genomic DNA of the tree shrew.An analysis of a wild population of 117 individuals collected from the suburb of Kunming,China,showed that these loci exhibited a highly expected heterozygosity(0.616).These 12 microsatellites were sufficient for individual identification and parentage analysis.The microsatellite markers developed in this study will be of use in evaluating genetic diversity and lineage tracing for the tree shrew.

Using SSR Marker to Trace Chinese Shrimp Fenneropenaeus chinensis Released in Natural Sea-A Feasible Strategy for Assessment of Release Effect in Natural Resources Recovery Program

Enhancement release has been proven effective in natural resources recovery of Chinese shrimp Fenneropenaeus chinensis in the last several decades in China, however, to assess the effectiveness of enhancement release, we still need to develop a high-efficient and easy-operational method to replace those physical-tagging release method with labor intensive, size-and number-limited. In the present study, single(with maternal known) parentage identification using eight simple sequence repeat(SSR) markers genotype fingerprint was used to trace Chinese shrimp released in Bohai Bay in 2013. A total of 884 shrimp spawners were collected from two hatcheries in Tianjin City respectively after enhancement release of shrimp larvae in May 2013. A total of 844 shrimp samples were recaptured around the release location approximately 4 months after the shrimp larvae were released into the natural sea. Genotype data of 8 SSR loci of the 1,726 samples were used for maternal-offspring parentage identification using CEervus 3.0 software. The allele number in each locus ranged from 8 to 68 with an average value of 33.25, which produced the cumulative exclusion probability with one parent known of all these sight loci up to 99%. Among the 844 recaptured shrimp samples, 448(♂:♀=212:232, gender information was lost for 4 samples) were successfully traced to their 337 maternal parents using a logarithm of odds(LOD) > 3.0 threshold. Among these 337 maternal parents, 253 had a single offspring, 62 had two offspring, 18 had three offspring, 3 had four offspring, and 1 had five offspring. For the first time, a large number of released shrimp were identified from recapture samples, and this study showed that it is possible to trace all released Chinese shrimp without using any type of physical tag in enhancement release activities. This not only means more precise recapture ratio assessment than ever expected, but also this method demonstrates an effective method for large-scale hatchery release as well as for organisms used in hatchery enhancement which are not suitable for physical tagging.

Development of two microsatellite multiplex PCR systems for high throughput genotyping in Populus euphratica

Eighteen microsatellite primer pairs previously developed at Oak Ridge National Laboratory for Populus tremuloides Michx. and Populus trichocarpa Torr. & Gray were screened for amplification in Euphrates poplar, Populus euphratica Oliv. Thirteen loci were found to express polymorphisms ranging from two to 17 alleles. The eight most variable loci were selected to set up and optimize two multiplex polymerase chain reaction (PCR) assays. Three populations containing altogether 436 trees were used to characterize the selected loci and ascertain their applicability for parentage analysis and genotyping studies. Through cross-checking of clonal identity against sex of the genotyped trees we estimated the maximum error rate for merging genotypes to be less than 0.045.

Reducing the Common Environmental Effect on Litopenaeus vannamei Body Weight by Rearing Communally at Early Developmental Stages and Using a Reconstructed Pedigree

Heritability estimates may be severely biased when a large common environmental effect on a family arises from a longlasting separate rearing at early stages(SRES)in traditional selective breeding programs,especially when bred populations have weak genetic ties.Communal rearing at early stages(CRES)may reduce common environmental effect since all families are reared in the same environment immediately after hatching.Here,we compared the effects of CRES and SRES strategies on genetic parameter estimation for harvest body weight in a selective breeding population of Litopenaeus vannamei with a small number of half-sib families.Genetic parameters of each strategy were estimated by using animal models excluding and including the common environmental effect(Model 1 and Model 2,respectively).Heritability estimates for body weight were 0.21±0.06(P<0.05)and 0.69±0.09(P<0.05)for CRES and SRES,respectively,in Model 1,and 0.21±0.06(P<0.05)and 0.52±0.27(P>0.05)in Model 2.The ratio of common environmental variance to phenotypic variance was 0.002±0.000 and 0.071±0.112 for CRES and SRES,respectively.Neither strategy precisely partitioned the common environmental variance according to likelihood ratio test.Lower heritability for body weight in CRES than in SRES implied that a large common environmental variance was confounded with additive genetic variance and was not effectively partitioned in SRES.Moreover,genetic correlation of body weight between the two strategies was 0.75±0.15,indicating that family rankings truly changed.The CRES should be followed in the selective breeding program of shrimp,especially in a population with a shallow pedigree and weak genetic ties between families.

WAVEFUNCTIONS OF L-S COUPLING FERMION SYSTEM(Ⅰ): SINGLE-l

In this paper. it is discussed how to constrnct wavefunctions of L-S couplingfermion system, which are classified by group chain A recurrent formula of fractional parentage coefficients with fixedseniority is also given.

WAVEFUNCTIONS OF L-S COUPLING FERMION SYSTEM (Ⅱ):DOUBLE■-l

The wavefunctions of L-S coupling fermion system, which are classified by group chain U(4ι1 + 4ι2 + 4) Us(2)×(U L(2ι+ 2ι2 + 2) O(2ι1 + 2ι2 + 2) O(2ι+1) ×O(2ι2 + 1) O1 (3)×O2 (3) O(3)), are constructed through introducing generalized pairs coupled by fermions with different ι. With the help of the fractional parentage coefficients of single-ιfermion system, the author obtains the corresponding fractional parentage coefficients of double-ιfermion system.

PCR技术在法医生物学中的应用

遗传学认为,DNA是人体性状及遗传变异的物质基础,它是遗传信息的携带者.DNA决定着我们的肤色、性别、身高、健康状况等个体特征.每个人的DNA都是不完全一样的.在法医学上,往往需要鉴别现场留下的物证是否属于犯罪嫌疑人.传统的方法主要是通过指纹、脚印、唇纹、化学物质等物证进行核对,这在一些情况下能起到作用.然而,有时候犯罪分子相当狡猾,或者现场被破坏的很严重,根本找不到上述痕迹.但是在理论上,只要犯罪分子在现场留下血渍、精斑、头发、阴毛、唾液等,通过分析其中的DNA,仍可查获真凶.另外,也可通过分析在现场找到的死者的残留物(如毛发、牙齿、骨骼等)中所含的DNA,来验明他们的真实身份.以前由于这些物证中DNA的含量甚微,再加上无法排除在分析时受到的干扰,DNA分析还或多或少是一种幻想.直到1985年,诺贝尔奖获得者Kary mullis发明了聚合酶链反应(polymerasechain reaction,PCR).再加上1988年Saiki对PCR的重大改进,才使DNA分析在法医学领域的应用取得了惊人的突破.所谓PCR,就是通过特定的DNA序列作为引物,在酶的作用下,经过几十次循环,使目的DNA得以快速复制、扩增,从而得到足以进行检测和分析的目的DNA片段.目的DNA(或称模板DNA)就好象是“轮子”,引物好象有汽车的“方向”,酶好象是“(?)